Chronic pathological pain and certain epileptic syndromes are neuropsychiatric disorders that share an increased female prevalence and refractoriness to treatment. The latter feature is considered to be linked to pathologically increased neuronal excitability caused by increased neuronal chloride (Cl-), which in turn is rooted in down-regulation of the dominant neuronal Cl--transporter, KCC2, which extrudes Cl-. Here we propose experiments to elucidate sex-specific regulation of the kcc2 gene by estrogens, based on a hypothesis that neuronal Cl- is dysregulated in response to neuronal injury in a sexually dimorphic manner, with the consequence of rendering women more susceptible to the above diseases. We have obtained exciting preliminary results (1) showing that kcc2 transcription is regulated by the repressor REST/NRSF which binds to a novel RE1/NRSE DNA binding site in kcc2 regulatory regions, (2) demonstrating this regulation to underlie the early developmental transformation of GABAergic transmission from excitatory to inhibitory, (3) developing a novel method to culture cortical primary neurons from individual rat E17 embryos which are being sex-typed by X-and Y-chromosome specific DNA markers. The latter method, straightforward yet possibly a groundbreaking novelty, permits strictly separate female vs. male primary cortical neuronal culture. We intend to elaborate molecular mechanisms how neuronal Cl- and KCC2 are regulated sex-specifically by exposing male vs. female neurons to 17-[unreadable]-estradiol and xenobiotic estrogen-mimetics. For this, we will electroporate kcc2 reporter gene constructs, wildtype and mutated for binding sites, driving a secreted luciferase reporter, which will facilitate establishment of a time-course of kcc2 transcription. For direct determination of Cl-, the fluorescent Cl--indicator clomeleon will be co-transfected. Cultures will be exposed to physiologically relevant concentrations of estradiol and practically relevant concentrations of xeno-estrogens (coumestrol, bisphenol-A, dieldrin). Use of the latter compounds will allow us to address modulation of estrogen responses by these ubiquitous compounds. Any sex-specific regulation will be confirmed in primary cultures derived from gene-targeted mice (estrogen-receptor (ER)-a, -[unreadable] and non-classical-ER-knockin). These experiments will be conducted in a highly collaborative environment at Duke University, involving molecular and physiology neuroscience labs, in addition molecular endocrinology and environmental toxicology input. Results can be expected to shed new light on a fundamental matter, neuronal Cl--regulation, which very likely has sex-specific regulation as a basis for increased female prevalence in therapy-refractory neuropsychiatric diseases. PUBLIC HEALTH RELEVANCE: Neuronal chloride dictates nerve cells'excitability, and is reduced in chronic pathological pain as well as in certain forms of epilepsy, diseases characterized by therapeutic refractoriness and strong female preponderance. Experiments are described that will elucidate the regulation of the dominant electroneutral chloride transporter of mature neurons, KCC2. Estrogen and xenobiotic estrogen-mimetics will be used for stimulation of primary cortical neurons in culture, which will be maintained strictly separate for male vs. female, based on a novel methodology platform described here. Neurons derived from late-pregnancy embryos of rats and mice, the latter genetically encoded to lack functional estrogen-receptors, will be subjected to assays probing function and regulation of the kcc2 gene, namely reporter gene assays and measurement of neuronal chloride.